PROJECT SUMMARY Chronic wounds such as venous leg ulcers (VLU), pressure sores and diabetic foot ulcers are challenging clinical problems that affect a growing number of people due to the global expansion of the elderly, diabetic and obese. VLU affect approximately 1% of the adult US population, i.e., 2.3 million individuals. There are approximately 1 million pressure ulcers per year in US with greatest impact on the elderly and spinal cord injured persons. Moreover, about 12 million individuals have diabetes mellitus in the US and it is expected that 25 % of these people will develop leg or foot problems. These wounds are difficult to treat and often drastic operative interventions such as amputations or free flaps with clear loss of function are necessary. The long term goals of this application are to develop novel therapies for healing of chronic wounds resulting because of diabetes or some other circulatory condition. Researchers have shown for years that proteins called growth factors improve the healing of chronic wounds, but their use in clinics is limited. This is commonly attributed to the delivery of soluble growth factors to the injury site which is expensive and challenging. However, animal models also lack the harsh protease rich environment present in human chronic wounds. The impact of this environment on growth factor delivery systems has not been examined. We hypothesize that it is the critical barrier to the successful translation of GF therapies to human patients. Here, we describe an innovative two pronged strategy to overcome this barrier. First, we propose to develop multifunctional nanoparticles (mNPs) comprised of GFs and protease inhibitor peptides (PIP) fused to elastin-like-peptide (ELPs). Elastin-like-peptides (ELPs) are attractive carriers owing to their biocompatibility and unique self- assembling properties. Recently, we have shown that chimeric fusion of GF and ELPs self-assembles into NPs without loss of biological activity of the GF. Based on this finding we hypothesize that addition of PIP-ELP to GF-ELP fusion protein will form mNPs that not only effectively deliver the GF but also protect it from proteolytic degradation. This mNP formulation will improve chronic wound healing outcomes over simply mixing the ingredients by ensuring the proximity of PIP with the GF. Second, we propose a novel animal wound model that has protease levels similar to human chronic wounds. We have preliminary data that correlates high protease activity with non-healing wounds in mice. We propose to use this model to test the ability of mNPs to heal the wounds. Successful completion of the project will result in protease resistant growth factor formulations that will induce healing in a proteolytic environment similar to human chronic wounds. Also, this research will shed light on the importance of proteases in wound healing and how excessive protease activity leads to a chronic wound. This approach can be easily extended to incorporate any growth factor and any protease inhibitor thereby broadening its scope beyond healing of chronic skin wounds.